*3.3. Metabolic Enzymes and Pathways*

Going deeper, we performed an in-silico pathway reconstruction using the detected proteins in the *L. infantum* promastigote proteome. By using the KEGG database resource (http://www.genome.jp/ kegg/) accessed via the DAVID package, a total of 578 (27.6%) of the detected proteins could be classified into pathways representing classical cellular processes. In particular, 236 proteins were identified as metabolic enzymes; 31% of these enzymes belong to glycolysis (Tables 1 and 2), the tricarboxylic acid (TCA) cycle, and the pentose phosphate cycle (Supplementary File, Table S4), which are three metabolic pathways playing essential maintenance functions in the cell [47]. Remarkably, the complete set of 29 enzymes that make up the TCA cycle were identified in the promastigote proteome (Figure 3).


**Table 1.** List of glycosomal enzymes related to gluconeogenesis and glycolysis identified in *L. infantum* (according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database).

**Table 2.** List of cytosolic enzymes related to gluconeogenesis and glycolysis identified in the *L. infantum* proteome (according to the KEGG pathway database).


The glycosome is a trypanosomatid-specific, membrane-enclosed organelle that contains glycolytic enzymes, among others. Thus, glycolysis in *Leishmania* takes place in these organelles for the steps between glucose and 3-phosphoglycerate [48], as well as in the cytosol for those late steps leading to the formation of pyruvate [20]. The identified enzymes involved in these two stages are listed in Tables 1 and 2. Among them, there are 32 enzymes belonging to the glycosomal/cytosolic glycolysis (and gluconeogenesis) pathway until the formation of pyruvate by pyruvate kinases (IDs LINF\_350005400 and LINF\_350005300). Some enzymes involved in the mitochondrial electron transport respiratory chain were detected (Supplementary File, Table S5). Similar findings were found by Rosenzweig and collaborators in the *L. donovani* promastigote proteome [20]. Several proteins of the electron transport chain are encoded by the kinetoplast DNA maxicircles [49] such as cytochrome oxidase subunits and NADH dehydrogenase, but they were not searched in this study.

An active energy metabolism requires enzymes to be involved in redox homeostasis. Several of these enzymes have been identified in the *L. infantum* proteome, and they are likely abundant, as judged by the large number of unique peptides that were mapped to them. The detected proteins were tryparedoxin (LINF\_150019000, with 31 unique peptides), peroxidoxin (LINF\_230005400, 31 peptides), cyclophilin (LINF\_060006300, 20 peptides), iron superoxide dismutases (LINF\_080007900 and LINF\_320024000, with 14 and 18 peptides, respectively), and several elongation factors 1β (LINF\_340014200 and LINF\_340014000 with 16 peptides each and LINF\_360020500 with 19 peptides).

**Figure 3.** Detected enzymes in the *L. infantum* JPCM5 proteome composing the complete tricarboxylic acid (TCA) cycle. PDH: pyruvate dehydrogenase; ACO: aconitase; IDH: isocitrate dehydrogenase; ODH: 2-oxoglutarate dehydrogenase; SL: succinyl-CoA ligase; SDH: succinyl dehydrogenase; FH: fumarate hydratase; MDH: malate dehydrogenase; and CS: citrate synthase.
